1. Field of the Invention
This invention relates to methods of determining the growth hormone status of an individual. This is performed by measuring the circulating Insulin-like Growth Factor binary or ternary complex (150 KDa complex containing IGF, IGFBP and ALS). The complex is first captured with a monoclonal antibody that is coupled to a solid support and called a "capture-antibody" herein. One of the members of the complex is detected with a monoclonal antibody called a "detection-antibody" herein.
Ideally, the capture- and detection-antibodies do not interfere with each other or with complex formation. For this reason, we have prepared a series of monoclonal antibodies and mapped their epitopes. The invention also pertains to said monoclonal antibodies and kits containing the same.
2. Background
The super family of insulin-like growth factors (IGFs) includes IGF-I and -II, at least six high affinity IGF binding proteins (IGFBP-1 to IGFBP-6), an acid-labile protein subunit (ALS), and various cell surface receptors, proteases and antagonists. The IGF superfamily is intimately involved in the regulation of cellular growth and metabolism (1-4). IGFs are produced by multiple normal and malignant tissues and are present in blood and other biological fluids in close association with IGFBPs which specifically bind and modulate their bioactivities (4-9). IGF-I, a 7.5 KDa peptide, is the major mediator of the in vivo mitogenic and metabolic effects of growth hormone (GH) and, as with IGF-II, its actions are mediated by both endocrine and autocrine-paracrine mechanisms (1-4).
Most of the IGF circulates with an approximately 150 KDa ternary protein complex consisting of IGFBP-3 and ALS (4-9). Binding of ALS to IGFBP-3 depends on IGFBP-3 occupancy by IGF-I or IGF-II and under normal conditions, nearly all of the circulating IGFs (&gt;95%) are found in the ternary complex (4-12). IGFBP-3 is normally the major carrier of IGF in serum and its association appears to stabilize daily IGF levels (half-life of .about.12-15 hrs) and limit IGF access to extracellular compartments (4-9). It is presumed that most IGFs are found in the ternary complex because ALS is present at significantly higher molar excess than IGFBP-3 or the IGF peptides (13, 14). Smaller proportions of circulating IGFs are associated with other IGF binding proteins and less than 1% has been estimated to exists in an unbound or "free" form.
While the precise biological relevance of the ternary complex has not been clearly defined, circulating levels of its constituents, and thus formation of the ternary complex, is highly GH-dependent (4-9). Serum IGF-I, IGFBP-3 and ALS are low in GH deficiency and are elevated in acromegalic subjects (4-9, 13, 15). Post-translational modifications of IGFBPs, including proteolysis (16, 17) and phosphorylation (18, 19), are apparently involved in modulating bioavailability of the IGF peptides.
The clinical assessment of GH status has been controversial, primarily due to the episodic nature of GH secretion, its relatively short circulating half-life and considerable variability in GH measurement by different methods (20, 21). Currently, clinical evaluation of GH sufficiency may involve multiple venous blood sampling for determination of GH secretion in response to a number of physiological or pharmacological stimuli. Because of the reported limitations of provocative GH testing, which include arbitrary definition of diagnostic cut-off levels, and potential health risk and cost, alternative screening procedures have been sought (20, 21).
As blood IGF-I, IGFBP-3 and ALS levels are highly dependent on GH secretion (4-9, 13, 15), appropriate determination of their serum levels have been endorsed as the most effective means in the evaluation of GH-IGF axis status, particularly in children with short stature (21-23). Determinations of IGF-I and ALS may be also valuable in the diagnosis of adult GH deficiency and monitoring of therapeutic response to GH replacement therapy (24, 25).
The high levels of association with IGFBPs has been a major concern in routine immunoassay of serum IGF-I because IGFBPs may mask reactive epitopes or compete with antibodies for tracer binding. Reliable determination of IGF-I in serum requires dissociation and removal of IGFBPs before analysis (26). Present strategies are based on serum acidification to irreversibly denature ALS and disrupt the ternary complex, followed by procedures to remove most or all of the IGFBPs. Size exclusion chromatography in acid is considered the "gold standard" method for the latter step (27), but is not practical for efficient high-volume sample processing.
The most commonly used alternative is acid-ethanol precipitation (28). However, the method may leave substantial residual amounts of IGFBPs (19, 29-32). We recently confirmed that IGFBPs, in particular low molecular weight IGFBPs such as IGFBP-1, may be retained in significant quantities after acid-ethanol precipitation (19). The residual amounts of IGFBPs could compete with the detection-antibody, particularly in competitive RIAs (29) where the amount of antibody and labelled IGF-I is small relative to the concentration of analyte and residual IGFBPs. This phenomenon may account for the well known propensity of IGF-I RIAs to give false estimates of IGF-I levels in samples with high levels of endogenous IGFBPs (27, 29, 30, 32).
Various means have been devised to minimize the interference of residual IGFBPs, including addition of IGF-II to the assay mixture, acid-ethanol cryoprecipitation, use of analog tracers with low affinity for IGFs or acid-Bio-Spin chromatography (29, 30, 32, 33). However, these methods further add to the complexity of the assay and may not completely resolve the IGFBP-interference problem.
The clinical assessment of IGF-I, IGFBP-3 and ALS is further complicated by the differential effects of age, pubertal stage of development and nutritional factors on their circulating levels (21, 34, 35). Accordingly, current recommendations suggest use of IGF-I, IGFBP-3 and ALS in younger age group, and IGF-I and ALS in adult subjects (21-26, 36). As nearly all of serum IGF-I, IGFBP-3 (4-12) and as much as 50% of ALS (13, 15) are present in the high molecular weight IGFBP-3 ternary protein complex, direct determination of the complex may represent an ideal and potentially superior screening alternative. However, until now, no one has presented a reliable, accurate and simple means of quantifying the level of IGFBP complex.